Class V myosins (myoV) play crucial roles in actin-based organelle transport and membrane trafficking, and are needed for survival from yeast to mammals. There are three isoforms in mammals, with different tissue specificities and functions. MyoVa is processive, meaning that the motor can take multiple steps on actin before dissociating. It has been implicated in transport of melanosomes, insulin granules, and endoplasmic reticulum. In contrast, myoVc has been reported to be non-processive and expressed in glandular tissue where it associates with large exocrine granules. Mutations in myoV lead to human disease (e.g. Griscelli syndrome, microvillus inclusion disease), attesting to their important role in normal cellular function. Kinesin and dynein play similar transport role on microtubule tracks. Their dysfunction causes a myriad number of neurodegenerative diseases. A broad theme of this proposal is to systematically build complexity into the in vitro study of cargo-transport motors to probe mechanisms. Each aim is motivated by biological observations at the cellular level. In Aim 1, through the use of full- length myoVa, native adaptor proteins, and tracks composed of cytoplasmic actin with bound tropomyosin, we investigate mechanisms involved in cargo localization by class V myosin motors. Single- molecule approaches and total internal reflection fluorescence (TIRF) microscopy will be used. In Aim 2, we investigate the properties of myoVc, the isoform which transports large secretory granules in exocrine tissues. We wish to understand how actin track morphology can be a processivity factor for myoVc, and how the ensemble properties of myoVc differ from those of myoVa, using DNA origami to couple multiple motors. In Aim 3 we will reconstitute a minimal mRNP containing full-length kinesin and dynein, adaptor proteins, and mRNA, to test ideas of motor co-ordination, activation, and directionality. As a whole, the proposed studies provide an essential bridge between well-defined single motor studies with truncated constructs, and purely in vivo cell biological studies of myosin, kinesin, and dynein motors.